The invention relates to certain atropisomer forms of asymmetric xanthene fluorescent dyes and the field of nucleic acid sequencing and analysis with fluorescent dye-labelled reagents.
Methods of analyzing fluorescent-labelled biomolecules after separating based on size- or charge are central to molecular biology. Examples of methods utilizing fluorescent-labelled nucleic acids include automated DNA sequencing, oligonucleotide probe methods, detection of polymerase-chain-reaction products, immunoassays, and the like. In the case of multi-color automated DNA sequencing, labelled nucleic acid fragments of varying size are separated by electrophoresis, typically in a single electrophoresis lane, channel, or capillary. Employing these methods, automated four-color Sanger-type DNA sequencing has enabled entire genome characterization at the molecular level.
Stereochemical purity is of importance in the field of pharmaceuticals, where 12 of the 20 most prescribed drugs exhibit chirality (U.S. Pat. No. 6,075,024). A case in point is provided by the L-form of the beta-adrenergic blocking agent, R(xe2x88x92) albuterol, which is known to be 100 times more potent than the D-enantiomer (U.S. Pat. No. 5,760,090). Furthermore, optical purity is important since certain isomers may actually be deleterious rather than simply inert.
Atropisomers are stereoisomeric conformations of a molecule whose interconversion is slow enough to allow separation and isolation under predetermined conditions (McGraw-Hill Dictionary of Chemical Terms, (1984), S. Parker, Ed., p. 36). The energy barrier to thermal racemization may be determined by the steric hindrance to free rotation of one or more bonds forming a chiral axis. Certain biaryl compounds exhibit atropisomerism where rotation around an intraannular bond lacking C2 symmetry is restricted. The free energy barrier for enantiomerization is a measure of the stability of the intraannular bond with respect to rotation. Optical and thermal excitation can promote racemization, dependent on electronic and steric factors (Tetreau (1982) Nouv. Jour. de Chimie, 6:461-65).
Ortho-substituted biphenyl compounds may exhibit this type of conformational, rotational isomerism known as atropisomerism (Eliel, E. and Wilen, S. (1994) Stereochemistry of Organic Compounds, John Wiley and Sons, Inc., pp. 1142-55). Such biphenyls are enantiomeric, chiral atropisomers where the sp2-sp2 carbon-carbon, intraannular bond between the phenyl rings has a sufficiently high energy barrier to free rotation, and where substituents Xxe2x89xa0Y and Uxe2x89xa0V render the molecule asymmetric. The steric interaction of Xxe2x80x94U, Xxe2x80x94V, and/or Yxe2x80x94V, Yxe2x80x94U is large enough to make the planar conformation an energy maximum. Two nonplanar, axially chiral enantiomers, shown below, then exist as atropisomers when their interconversion is slow enough such that they can be isolated free of each other. By one definition, atropisomerism is defined to exist where the isomers have a half-life txc2xd of at least 1000 seconds, which is a free energy barrier of 22.3 kcal molxe2x88x921 (93.3 kJ molxe2x88x921) at 300K (Oki, M. (1983) xe2x80x9cRecent Advances in Atropisomerism,xe2x80x9d Topics in Stereochemistry, 14:1). Bold lines and dashed lines in the figures shown below indicate those moieties, or portions of the molecule, which are sterically restricted due to a rotational energy barrier. Bolded moieties exist orthoganally above the plane and dashed moieties exist orthogonally below the plane of the rest of the molecule. 
Xanthene dyes have important applications as detectable fluorescent labels of nucleic acids (U.S. Pat. Nos. 5,188,934; 5,654,442; 5,885,778; 6,096,723; 6,020,481; 5,863,727; 5,800996; 5,945,526; 5,847,162; 6,025,505; 6,008,379; 5,936,087; 6,015,719). Xanthene compounds containing an asymmetric biannular bond can exist in stable atropisomeric forms. Conjugates of atropisomeric xanthene compounds and chiral substrates, such as nucleotides, polynucleotides, polypeptides, and carbohydrates, form diastereomers. These diastereomeric conjugates can separate under certain conditions, such as electrophoresis, chromatography, and other methods. Separation of diastereomers can hinder detection by display of double peaks or bands, i.e. xe2x80x9cpeak doublingxe2x80x9d. Thus, atropisomerically enriched or purified forms of xanthene dyes are important as labels for methods based on separation and detection of analytes.
The present invention is directed towards atropisomerically-enriched and substantially pure atropisomers of asymmetric xanthene compounds as novel compositions. The invention also includes methods for isolation, labelling, and detecting labelled compositions.
In a first aspect, the invention includes substantially pure atropisomer compounds having the structure II: 
wherein positions R1, R4, R5, R11, R13, R14, R17, R18, R19, R20, Z1, or Z2may be substituted with substituents. At least one substituent may be a linking moiety. One or more rings may be fused on the ring structure II.
Another aspect of the invention includes energy-transfer dye compounds comprising a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response thereto; an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and a linker for linking the donor dye and the acceptor dye; wherein at least one of the donor dye and acceptor dye is a substantially pure atropisomer of a xanthene compound.
Another aspect of the invention is labelled substrates, including nucleoside, nucleotides, polynucleotides, and polypeptides wherein the label is a substantially pure atropisomer of a xanthene compound or an energy-transfer dye comprising a substantially pure atropisomer of a xanthene compound.
Another aspect of the invention is labelling reagents, including phosphoramidite and active ester linking moieties of a substantially pure atropisomer of a xanthene compound, which form covalent attachments with substrates and methods of labelling substrates with the reagents.
Another aspect of the invention is methods for forming a labelled substrate comprising the step of reacting a substrate with the linking moiety of a substantially pure atropisomer of a xanthene compound or an energy-transfer dye comprising a substantially pure atropisomer of a xanthene compound.
Another aspect of the invention is methods for separating atropisomers of xanthene compounds by forming diastereomers with substantially enantiomerically pure compounds, and separating the diastereomers. The diastereomers may be converted to substantially pure atropisomers of xanthene compounds.
Another aspect of the invention is methods for separating a mixture of labelled substrates wherein the labels are comprised of a substantially pure atropisomer of a xanthene compound or an energy-transfer dye comprising a substantially pure atropisomer of a xanthene compound. The labelled substrates may be primer extension polynucleotide fragments. The labelled substrates may be separated by electrophoresis, chromatography, or other separation technique. The mixture of labelled polynucleotides may be formed from a labelled primer or a labelled terminator. The labelled substrates may be detected by fluorescence detection.
Another aspect of the invention is methods of generating a labelled primer extension product by extending a primer-target hybrid with an enzymatically-incorporatable nucleotide. The primer or the nucleotide may be labelled with a substantially pure atropisomer of a xanthene compound or an energy-transfer dye comprising a substantially pure atropisomer of a xanthene compound.
Another aspect of the invention is methods of polynucleotide sequencing by forming a mixture of four classes of polynucleotides where each class is labelled at the 3xe2x80x2 terminal nucleotide with a substantially pure atropisomer of a xanthene compound or an energy-transfer dye comprising a substantially pure atropisomer of a xanthene compound, and the labels are spectrally resolvable. The polynucleotides are separated by size.
Another aspect of the invention is methods of oligonucleotide ligation by annealing two probes to a target sequence and forming a phosphodiester bond between the 5xe2x80x2 terminus of one probe and the 3+ terminus of the other probe wherein one or both probes are labelled with a substantially pure atropisomer of a xanthene compound or an energy-transfer dye comprising a substantially pure atropisomer of a xanthene compound.
Another aspect of the invention is methods of amplification by annealing two or more primers to a target polynucleotide and extending the primers by a polymerase and a mixture of enzymatically-extendable nucleotides wherein at least one of the primers or one of the nucleotides is labelled with a substantially pure atropisomer of a xanthene compound or an energy-transfer dye comprising a substantially pure atropisomer of a xanthene compound.
Another aspect of the invention is kits of reagents including a substantially pure atropisomer of a xanthene compound or an energy-transfer dye comprising a substantially pure atropisomer of a xanthene compound.